In the early days of bone marrow transplantation (BMT), many transplants failed because of rejection of the graft by the host. Transplants that did succeed, however, often led to an immune response by lymphocytes present in the graft against various tissues of the host (Graft versus Host Disease “(GvHD)”). It is now known that the GvHD response is mainly due to the presence of major histocompatibility (H) antigens which present a transplantation barrier. Therefore, it is now routine practice to graft only HLA-matched materials (either from siblings or unrelated individuals) resulting in a much improved rate of success in bone marrow transplantation. However, despite this improvement, as well as improvements in pre-transplantation chemotherapy or radiotherapy and the availability of potent immunosuppressive drugs, about 20–70% of the treated patients still suffer from GvHD (the percentage is age and bone marrow donor dependent). To avoid GvHD, it has been suggested to remove the cells (mature T cells) causing the reaction from the graft. This however often leads to graft failure or to recurrence of the original disease. The cells responsible for GvHD are also the cells which often react against the original aberrant cells in, for instance, leukemia (Graft versus Leukemia response).
Since BMT is now mainly carried out with HLA matched grafts, the GvHD which still occurs must be caused by another group of antigens. It is very likely that the group of so-called minor H antigens (mHag), which are non-MHC encoded histocompatibility antigens (unlike the major H antigens) are at least partially responsible for the remaining incidence of GvHD. mHag's have originally been discovered in congeneic strains of mice in tumor rejection and skin rejection studies. In mice, the use of inbred strains has shown that mHag are encoded by almost 50 different allelically polymorphic loci scattered throughout the genome. In humans, although cumbersome to identify, mHag have been shown to exist, but their overall number and complexity remains uncertain. Minor H antigens are most likely quite different from each other and quite different from major H antigens, they are probably a diverse and elusive group of fragments of molecules which are participating in various cellular housekeeping functions. Their antigenicity may come very incidentally, as naturally processed fragments of polymorphic proteins that associate with MHC products. Some of the mH antigens appear to be widely expressed on various tissues throughout the body whereas others show limited tissue distribution.
One of the better known minor histocompatibility antigens is the H-Y antigen. H-Y is a mHag that can lead to rejection of HLA-matched male organ and bone marrow grafts by female recipients, and to a higher incidence of GvHD in female-to-male grafts, particularly if the female donor had been previously pregnant. The H-Y antigen may also play a role in spermatogenesis. The human H-Y antigen is an 11 residue peptide derived from SMCY, an evolutionary conserved Y chromosomal protein. Another well known mHag that can lead to GvHD is the HA-2 antigen. The human HA-2 antigen is a 9 residue peptide likely derived from a class I myosin. However, the nature of the HA-1 antigen, responsible for a majority of current cases of GvHD has remained elusive so far.
Human bone marrow transplants performed as therapeutical treatment of severe aplastic anemia, leukemia and immune deficiency disease became available in the seventies. For the present, the long-term results of allogeneic bone marrow transplantation (BMT) have greatly improved due to the use of HLA-matched siblings as marrow donors, advanced pretransplant chemoradiotherapy, the use of potent immunosuppressive drugs as Graft-versus-Host-Disease (GvHD) prophylaxis, better antibiotics and isolation procedures. Nonetheless, the results of clinical BMT reveal that the selection of MHC identical donors/recipients is not a guarantee of avoidance of GvHD or disease free survival even when donor and recipient are closely related. Allogeneic BMT especially in adults results, depending on the amount of T cell depletion of the graft, in up to 80% of the cases in GvHD. In the HLA genotypically identical situation it amounts to 15–35% whereas in the phenotypical HLA matched recipient/donor combinations, the occurrence of GvHD is significantly higher i.e. 50–80%.
Disparities for minor Histocompatibility antigens (mHag) between donor and recipient constitute a potential risk for GvHD or graft failure, which necessitate life long pharmacologic immunosuppression of organ and bone marrow transplant recipients. It is also believed that mHag are involved in the “beneficial” side effect of GvHD i.e. the Graft-versus-Leukemia activity. Several reports demonstrated the presence of anti-host mHag specific CTL in patients suffering from GvHD after HLA genotypically identical BMT. In our laboratory, much effort was put into the further characterization of a (small) number of anti-host mHag specific CTLS. Hereto, CTL clones specific for host mHag were isolated from the peripheral blood (PBL) of patients suffering from severe GvHD. mHag HA-1 specific CD8+ CTL clones were originally obtained after restimulation of in vivo primed PBLs from three patients suffering from GvHD after HLA identical but mHag nonidentical BMT. The post BMT CTL lines were cloned by limiting dilution, resulting in the isolation of a large number of mHag-specific CTL clones. Subsequent immunogenetic analyses revealed that the CTL clones (as described above) identified five non-sexlinked mHag, designated HA-1, -2, -3, -4, -5, which are recognized in a classical MHC restricted fashion. mHag HA-3 is recognized in the presence of HLA-A1 and mHag HA.-1, -2, -4 and 5 require the presence of HLA-A2.
Segregation studies demonstrated that each of mHag HA-1 to HA-5 is the product of a single gene segregating in a Mendelian fashion and that HA-1 and HA-2 are not coded within the HLA region. The mHag differ from each other in phenotype frequencies: mHag HA-1 appeared relatively frequent (i.e. 69%) whereas mHag HA-2 appeared very frequent (i.e. 95%) in the HLA-A2 positive healthy population. An inventory in five patients of mHag HA-1, -2, -3, -4 and -5 specific anti-host CTL responses after BMT demonstrated in 3 patients clones specific for the mHag HA-1. This observation points towards the immunodominant behavior of mHag HA-1. With regard to the mHag expressed on different tissues, we observed ubiquitous versus restricted tissue distribution of the mHag analyzed.
The expression of the mHag HA-1 is restricted to the cells of the hematopoietic cell lineage, such as thymocytes, peripheral blood lymphocytes, B cells, monocytes. Also, the bone marrow derived professional antigen presenting cells the dendritic cells and the epidermal Langerhans cells, express the mHag HA-1. The mHag HA-1 is also expressed on clonogenic leukemic precursor cells as well as on freshly isolated myeloid and lymphoid leukemic cells, indicating that mHag specific CTLs are capable of HLA class I restricted antigen specific lysis of leukemic cells.
To substantiate the importance of the human mH antigenic systems, we investigated whether the mHag are conserved in evolution between human and non human primates. Hereto, cells from non human primates were transfected with the human HLAA2.1 gene. Subsequent analyses with our human allo HLA-A2.1 and four mHag A2.1 restricted CTL clones revealed the presentation of ape and monkey allo and mHag HY, HA-1 and HA-2 peptides in the context of the transfected human HLA-A2.1 molecule by ape and monkey target cells. This implicates that the HA-1 peptide is conserved for at least 35 million years. A prospective study was performed in order to document the effect and clinical relevance of mHag in HLA genotypically identical BMT on the occurrence of acute (grade≧2) GvHD. The results of the mHag typing using the CTL clones specific for five well defined mHag HA-1 to HA-5 demonstrated a significant correlation between mHag HA-1, -2, -4 and -5 mismatch and GvHD. A significant correlation (P=0.024) with the development of GvHD was observed when analyzed for only mHag HA-1.
To analyze a putative peptidic nature of the mHag HA-1, we analyzed the requirement of the MHC encoded TAP1 and TAP2 gene products for mHag peptide presentation on the cell surface. The transporter genes TAP1 and TAP2 associated with antigen presentation are required for delivery of peptides from the cytosol with the endoplasmic reticulum. The availability of a human celline “T2” lacking both transport and proteasome subunit genes enabled us to study the processing and presentation of human mHag. We demonstrated that the (rat) transport gene products TAP1 and TAP2u were required for processing and presentation of antigenic peptides from the intracellular mH protein HA-1. Information on the TCR repertoire post BMT in man is extremely scarce. We have analyzed the composition of the T cell receptor (TCR) V region of mHag HA-1 specific CD8+ CTL clones by DNA sequencing of the α and β chains. We observed by analyzing TCR usage of 12 clones derived from 3 unrelated individuals that the TcRβ chains all used the TCRβV6S9 gene segment and showed remarkable similarities within the N-D-N regions.
However, until the present invention no one has succeeded in identifying amino acid sequences of antigenic peptides relevant to the mHag HA-1 antigen, nor has anyone succeeded in the identification of the proteins from which this antigen is derived. We have now for the first time identified a peptide which is a relevant part of mHag HA-1.